Division of Pharmacotherapy and Experimental Therapeutics, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, Kuopio, Finland.
Division of Pharmacotherapy and Experimental Therapeutics, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; DMPK Research Department, Teijin Pharma Limited, Hino, Tokyo, Japan.
Eur J Pharm Sci. 2019 Mar 15;130:186-195. doi: 10.1016/j.ejps.2019.01.022. Epub 2019 Jan 24.
Human-derived hepatic cell lines are a valuable alternative to primary hepatocytes for drug metabolism, transport and toxicity studies. However, their relevance for investigations of drug-drug and drug-organic anion (e.g., bile acid, steroid hormone) interactions at the transporter level remains to be established. The aim of the present study was to determine the suitability of the Huh7 cell line for transporter-dependent experiments. Huh7 cells were cultured for 1 to 4 weeks and subsequently were analyzed for protein expression, localization and activity of solute carrier (SLC) and ATP-binding cassette (ABC) transporters involved in organic anion transport using liquid chromatography-tandem mass spectroscopy, immunocytochemistry, and model substrates [H]taurocholate (TCA), [H]dehydroepiandrosterone sulfate (DHEAS) and 5(6)-carboxy-2',7'-dichlorofluorescein (CDF) diacetate. The extended 4-week culture resulted in a phenotype resembling primary hepatocytes and differentiated HepaRG cells: cuboidal hepatocyte-like cells with elongated bile canaliculi-like structures were surrounded by epithelium-like cells. Protein expression of OSTα, OSTβ and OATP1B3 increased over time. Moreover, the uptake of the SLC probe substrate DHEAS was higher in 4-week than in 1-week Huh7 cultures. NTCP, OATP1B1, BSEP and MRP3 were barely or not detectable in Huh7 cells. OATP2B1, MRP2 and MRP4 protein expression remained at similar levels over the four weeks of culture. The activity of MRP2 and the formation of bile canaliculi-like structures were confirmed by accumulation of CDF in the intercellular compartments. Results indicate that along with morphological maturation, transporters responsible for alternative bile acid secretion pathways are expressed and active in long-term cultures of Huh7 cells, suggesting that differentiated Huh7 cells may be suitable for studying the function and regulation of these organic anion transporters.
人源肝细胞系是进行药物代谢、转运和毒性研究的替代原代肝细胞的有价值的工具。然而，它们在研究转运体水平的药物-药物和药物-有机阴离子（例如胆汁酸、甾体激素）相互作用方面的相关性仍有待确定。本研究旨在确定 Huh7 细胞系是否适合进行依赖于转运体的实验。将 Huh7 细胞培养 1 至 4 周，然后使用液相色谱-串联质谱法、免疫细胞化学和模型底物 [H]牛磺胆酸钠（TCA）、[H]脱氢表雄酮硫酸酯（DHEAS）和 5(6)-羧基-2',7'-二氯荧光素（CDF）二乙酸酯分析涉及有机阴离子转运的溶质载体（SLC）和 ATP 结合盒（ABC）转运体的蛋白表达、定位和活性。扩展的 4 周培养导致表型类似于原代肝细胞和分化的 HepaRG 细胞：具有拉长的胆小管样结构的多角形肝细胞样细胞被上皮样细胞包围。OSTα、OSTβ 和 OATP1B3 的蛋白表达随时间增加。此外，在 4 周 Huh7 培养物中，SLC 探针底物 DHEAS 的摄取高于 1 周 Huh7 培养物。NTCP、OATP1B1、BSEP 和 MRP3 在 Huh7 细胞中几乎无法检测到或无法检测到。在 4 周的培养过程中，OATP2B1、MRP2 和 MRP4 的蛋白表达保持相似水平。通过在细胞间腔室中积累 CDF 证实了 MRP2 的活性和胆小管样结构的形成。结果表明，随着形态成熟，负责替代胆汁酸分泌途径的转运体在 Huh7 细胞的长期培养中表达并具有活性，这表明分化的 Huh7 细胞可能适合研究这些有机阴离子转运体的功能和调节。
